Biophysical studies of membrane proteins/peptides. Interaction with ...

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5274 Biochemistry, Vol. 45, No. 16, 2006 Fernandes et al. FIGURE 3: Absorption and fluorescence emission spectra of the indole class of V-ATPase inhibitors. Fluorescence emission spectra were obtained using excitation wavelengths corresponding to the maxima of each inhibitor: (thin dashed line) absorption spectrum of INH-1; (thin solid line) absorption spectrum of SB 242784; (thick dashed line) fluorescence spectrum of INH-1; (thick solid line) fluorescence spectrum of SB 242784. emission maximum was 432 nm. For the alternative incorporation method of adding the inhibitor to the preformed vesicles, the fluorescence emission maxima were slightly blue shifted (≈3 nm) when comparing with the previous values, but after a small period of time (≈5 min) they became identical to the ones obtained through the cosolubilization method, thus suggesting that the initially formed aggregates are disrupted upon membrane interaction. The quantum yields of SB 242784 and INH-1 determined in the presence of 2 mM DOPC LUVs were 0.15 and 0.10, respectively. These values are not biased by the fraction of inhibitors in the aqueous phase, because at this lipidic concentration it can be concluded from the determined partition coefficients (see below) that the inhibitors are almost completely incorporated in the membrane (>95%), and in addition, the quantum yield in water is negligible. Although an extremely large blue shift in the inhibitor fluorescence is observed upon incorporation in lipid vesicles (e.g., the maximum emission wavelength of SB 242784 in water is 505 nm), this is not per se evidence for a large extent of partitioning of the two molecules to the lipid environment, because, as mentioned above, the quantum yield in the aqueous medium is very low due to aggregation. To obtain a quantitative description, both the extent and kinetics of the partition of both inhibitors to DOPC vesicles were studied. Partition coefficients were determined through monitoring of the change in fluorescence emission intensity (I) vs lipid concentration [L] (Figure 4) and applying the equation (28): I ) I w + K p γ L [L]I L 1 + K p γ L [L] (5) where I w is the fluorescence of the fluorophore in water, I L is the fluorescence of the fluorophore incorporated in the lipid vesicles, K p is the partition coefficient defined in terms of solute concentrations in each phase, and γ L is the lipid molar volume [0.78 dm -3 mol -1 for DOPC (29)]. The results FIGURE 4: Increase in steady-state fluorescence emission intensity of indole V-ATPase inhibitors with lipid concentration: (b) SB 242784 incorporated in DOPC vesicles by the cosolubilization method; (O) INH-1 incorporated in DOPC vesicles by the method of addition after lipid solubilization. The buffer used was 10 mM Tris and 150 mM NaCl, pH ) 7.4. The lines represent the fits of eq 5 to the data, and the partition coefficients are shown in Table 1. Table 1: V-ATPase Inhibitor Partition Coefficients to Lipid Vesicles system inhibitor cosolubilization with DOPC cosolubilization with DOPC-DOPG addition to liposomes with DOPC SB 242784 (1.20 ( 0.08) × 10 4 (6.64 ( 1.76) × 10 3 (1.29 ( 0.23) × 10 4 INH-1 (1.49 ( 0.26) × 10 4 (2.84 ( 0.99) × 10 4 from a nonlinear fitting of eq 5 to the experimental data are shown in Table 1. For SB 242784, the anisotropy obtained while adding the inhibitor to preformed vesicles was 〈r〉 ) 0.33, the same value being obtained through the cosolubilization method. In vesicles composed of DOPC-DOPG (4:1 mol/mol), SB 242784 anisotropy was virtually identical (〈r〉 ) 0.32). The INH-1 anisotropy in samples with preformed vesicles (〈r〉 ) 0.24) was lower than observed for the same molecule incorporated by cosolubilization (〈r〉 ) 0.31). Again, these experiments were carried out at a lipidic concentration for which the fraction of light emitted from inhibitor molecules in water is negligible. Inhibitor TransVerse Location in Lipid Vesicles. The effects on the inhibitors’ fluorescence emission spectral shape and intensity caused by the presence of nitroxide-labeled lipid in DOPC vesicles were investigated. A 10% ratio of labeled (5- or 12-DOX-PC) to nonlabeled lipids was used. For SB 242784, there was a very slight shift on the fluorescence emission maximum, depending on the nitroxide-labeled lipid present. In the absence of quenchers the fluorescence emission maximum was at 436 nm, whereas in the presence of 5-DOX-PC this wavelength was 435 nm. Using 12-DOX- PC a red shift was obtained, the maximum being now 438 nm (Figure 5A). For INH-1, there was no difference in the shape of the emission spectrum in the presence of 12-DOX- PC (maxima were at 461 nm), but using 5-DOX-PC a very small blue shift (2 nm) was obtained (Figure 5B). These shifts are evidence that the two nitroxide labels, which are localized at different depths in the membrane, are selectively quenching inhibitor populations which lie at different transverse positions.
V-ATPase Indole Inhibitor Interaction with Bilayers Biochemistry, Vol. 45, No. 16, 2006 5275 FIGURE 6: Orientation of the transition moment of SB 242784, obtained from TD-DFT calculations (see text for details). Table 2: Order Parameters and Lagrange Coefficients of the Two Inhibitors in DOPC Multilayers Obtained from Linear Dichroism Studies a 〈P 2〉〈P 4〉 λ 2 λ 4 SB 242784 0.15 -0.21 0.99 -2.31 INH-1 0.18 -0.31 1.27 -4.67 a See text for details. FIGURE 5: Fluorescence emission spectra of indole V-ATPase inhibitors in the absence (s) and presence of different nitroxidelabeled lipids: (- - -) 5-DOX-PC and (‚‚‚) 12-DOX-PC). (A) SB 242784. (B) INH-1. Quantitative information on the inhibitor location in the membrane can be obtained via the so-called parallax method (30) using the equation: z cf ) L cl + (-ln(F 1 /F 2 )/πC - L 21 2 )/2L 21 (6) where z cf is the distance of the fluorophore from the center of the bilayer, F 1 is the fluorescence intensity in the presence of the surface-located quencher (5-DOX-PC), F 2 is the fluorescence intensity in the presence of the quencher located deeper in the membrane (12-DOX-PC), L c1 is the distance of the shallow quencher from the center of the bilayer, L c2 is the distance of the deep quencher from the center of the bilayer, L 21 is the distance between the shallow and deep quenchers (L c1 - L c2 ), and C is the concentration of the quencher in molecules/Å 2 . Using the distances of the nitroxide quenchers from the center of the bilayer given by Abrams and London (31), the obtained position for INH-1 was of 11.9 Å from the center of the bilayer, whereas for SB 242784 this value was 12.8 Å. To gain further information regarding the inhibitors’ location in the membrane, acrylamide quenching assays were also performed. Acrylamide is a hydrophilic quencher, and therefore differences in the extent of quenching by acrylamide allow a direct comparison of the bilayer penetration by the fluorophores. Acrylamide quenched both inhibitors with very similar efficiencies (results not shown), and a very slightly increased quenching observed for INH-1 is due to the also slight difference in average lifetimes (16) for the two inhibitors while incorporated in DOPC vesicles (〈τ〉 INH-1 ) 0.60 ns and 〈τ〉 SB 242784 ) 0.55 ns), because the Stern- Volmer rate constant is the product between the effective bimolecular rate constant (which can be decreased due to fluorophore shielding from the quencher) and the intrinsic lifetime of the fluorophore. Inhibitor Orientation in Lipid Vesicles. From the linear dichroism methodology, information regarding the orientation of the transition moment is obtained. In this way, to obtain information about the orientation of the molecule regarding the director of the system (normal to the membrane interface), it is necessary to know the orientation of the transition moment relative to the molecular axes. To this effect, a quantum chemical calculation was carried out. Applying time-dependent density functional theory (TD- DFT) to an energy-optimized geometry of SB 242784, the orientation of the transition dipole moment relative to the molecule structure was obtained. As shown in Figure 6, the transition dipole moment is almost parallel to the molecular axis defined by the double bond conjugated system, and an approximately identical transition dipole moment orientation can be considered for INH-1, as both molecules have very similar fluorophores and the molecular differences are not expected to largely influence this property. The orientation parameters 〈P 2 〉 and 〈P 4 〉 and Lagrange coefficients obtained for INH-1 and SB 242784 from the linear dichroism measurements are presented in Table 2. The corresponding orientational density probability functions are shown in Figure 7. DISCUSSION Inhibitor Aggregation and Partition to Lipid Vesicles. It is surprising that INH-1 appears to aggregate in aqueous solution more readily and to a larger extent than SB 242784. The presence of the piperidine ring in the latter was expected
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UNIVERSIDADE TÉCNICA DE LISBOA INS
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Fernandes, F., Loura, L. M. S., Fed
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Ao Pedro e Hugo, amigos de longa da
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2.2. - Peptides as models 2.3. - Am
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ABBREVIATIONS AND SYMBOL LIST ABBRE
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RESUMO RESUMO As biomembranas são
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SINOPSE SINOPSE Nas últimas duas d
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SINOPSE podem fornecer informação
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SINOPSE aceitantes. Dadores mais pr
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OUTLINE OUTLINE The last two decade
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OUTLINE membranes with a distributi
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OUTLINE BAR domains (tubulation of
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ion diffusion, as the energy requir
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acid. If no more groups are linked
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sphingomyelin, the most abundant sp
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signal for neighbouring cells to ph
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functional role in process such as
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in the L α phase, while lateral di
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1.7. Lateral heterogeneity in lipid
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the vesicle through bilayer deforma
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Figure I.9 - Depiction of the sever
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Figure I.11 - Experimentally obtain
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drying into a film and ressuspensio
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deactivating agents. Zwitterionic d
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amphipatic helices (see Section 2.3
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size corresponding to the hydrophob
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changes abruptly in the interfacial
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thickness of the bilayer (Section 1
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some α-helical membrane proteins a
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The formation of a lipid population
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Page 75 and 76: PROTEIN-PROTEIN AND PROTEIN-LIPID I
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Page 83 and 84: 2430 Biophysical Journal Volume 85
Page 85 and 86: 2432 Fernandes et al. Coat protein
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Page 95: QUANTIFICATION OF PROTEIN-LIPID SEL
Page 98 and 99: FRET Study of Protein-Lipid Selecti
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Page 111: INTERACTION OF THE INDOLE CLASS OF
Page 114 and 115: 5272 Biochemistry, Vol. 45, No. 16,
Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
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Page 140 and 141: Introduction Quinolones are broad-s
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Page 148 and 149: ⎛ II t ⎛ R ⎞ 0 ρ () t = exp
Page 150 and 151: APPENDIX - Derivation of the FRET r
Page 152 and 153: 2 2w J ( t) = '2 R − R + w / 1
Page 154 and 155: [14] L. Plançon, M. Chami, L. Lete
Page 156 and 157: acceptors) (Eqs. 4-6) (⋅-⋅-⋅)
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dynamin. Soon after, the same group
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140
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142
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Introduction Control of membrane re
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Steady-state fluorescence measureme
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Rho-DOPE (acceptor). The increase o
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where η is the viscosity of the me
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ound conformation of the BAR domain
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19. Fernandes, F., L. M. S. Loura,
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Figure Legends Fig.1: N-terminal am
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FIG. 1A FIG.1B 17
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FIG. 3A FIG. 3B 19
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FIG.5A 21
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FIG 6. 23
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FIG. 8 A B 25
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The signalling functions of PI(4,5)
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172
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174
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zoxadiazol (NBD)-PC were obtained f
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Fig. 3. Fluorescence decays of diph
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CONCLUSIONS VII CONCLUSIONS Chapter
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CONCLUSIONS constants recovered by
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CONCLUSIONS Chapter IV ‐ Binding
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CONCLUSIONS Chapter VI - Clustering
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FINAL CONSIDERATIONS AND PROSPECTS
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BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
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BIBLIOGRAPHY Phosphatidylinositol 4
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BIBLIOGRAPHY Glaubitz, C., Grobner,
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BIBLIOGRAPHY Koehorst, R. B. M., Sp
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BIBLIOGRAPHY Mishra, V. K., Palguna
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BIBLIOGRAPHY Pluschke, G., Hirota,
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BIBLIOGRAPHY Sperotto, M. M., and M
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BIBLIOGRAPHY Williamson, I. M., Alv
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